EP0177382A1 - Sonde mixte et ses applications, son précurseur, leur préparation et dérivés comportant une base modifiée pour cette préparation - Google Patents

Sonde mixte et ses applications, son précurseur, leur préparation et dérivés comportant une base modifiée pour cette préparation Download PDF

Info

Publication number
EP0177382A1
EP0177382A1 EP85401666A EP85401666A EP0177382A1 EP 0177382 A1 EP0177382 A1 EP 0177382A1 EP 85401666 A EP85401666 A EP 85401666A EP 85401666 A EP85401666 A EP 85401666A EP 0177382 A1 EP0177382 A1 EP 0177382A1
Authority
EP
European Patent Office
Prior art keywords
group
groups
probe
precursor
hydrogen atom
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP85401666A
Other languages
German (de)
English (en)
French (fr)
Inventor
Tam Huynh-Dinh
Béatrice Langlois d'Estaintot
Mario Zakin
Nathalie Duchange
Jean Igolen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Public Dit Centre National de la Ets
Institut Pasteur de Lille
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut Pasteur de Lille filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP0177382A1 publication Critical patent/EP0177382A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays

Definitions

  • the invention relates to oligonucleotide sequences which can replace mixed probes for the detection and isolation of nucleotide sequences.
  • nucleotide sequences in particular messenger RNA or complementary DNA, their precursors and their preparation. and their uses.
  • the subject of the invention is sequences of oligonucleotides of the above-mentioned type comprising modified bases, their preparation from precursors comprising bases which, by dismutation, lead to the modified bases in question, the preparation of these precursors, as well as the use of the probes obtained.
  • RNA messenger RNA by synthetic oligodeoxy-nucleotides, ie the detection, the isolation and the analysis of sequences.
  • messenger RNA by hybridization with complementary DNA fragments (cDNA). This technique is based on the pairing of the pairs A-T (2 hydrogen bonds) and G-C (3 hydrogen bonds).
  • the present invention is based on the discovery that it is possible to obtain, by dismutation of a single oligonucleotide sequence, under very specific conditions, a mixed sequence comprising, whenever there is an ambiguity in pyrimidine, an equimolecular amount of 5-methyl cytosine ( Me C ) and thymine (T) or an equimolecular amount of uracil (U) and cytosine (C), and whenever ambiguity arises in terms of '' a purine, an equimolecular amount of guanine (G) and 2-amino adenine ( NH 2 A ).
  • the mixed sequence thus obtained which contains certain modified bases Me C and / or NH2 A has all the characteristics of the mixed sequences prepared by the conventional means described above.
  • the subject of the invention is a probe precursor consisting of an oligonucleotide fragment of structure predetermined by the intended application, the reactive functions of which are optionally protected and which is optionally attached to a support, characterized in that at least part of the pyrimidine bases are replaced there by groups X and / or at least part of the purine bases there is replaced by groups Y, the groups X and Y corresponding respectively to the formulas and R representing a hydrogen atom or a methyl group.
  • the subject of the invention is a probe precursor consisting of an oligonucleotide fragment of structure predetermined by the envisaged application, the reactive functions of which are optionally protected and which is optionally attached to a support, characterized in that each ambiguity at the level of a pyrimidine base is replaced there by a group X and each ambiguity at the level of a purine base is replaced there by a group Y, the groups X and Y being as defined above.
  • the precursor according to the invention can be prepared according to any conventional process for the preparation of oligonucleotide fragments by replacing at least part of the pyrimidine bases with X groups and / or at least part of the purine bases with Y groups.
  • the preparation is carried out in the solid phase, essentially by fixing a monomer, dimer or trimer by its 3 'end to the 5' end of the 3 'terminal nucleotide of the desired sequence, fixed on a solid support, then fixations. successive monomers, dimers and / or trimers, each time on the terminal nucleotide 5 ′ of the chain obtained, until the desired oligonucleotide fraction is obtained.
  • the preparation of monomers, dimers and trimers carrying protective groups of this type can be carried out according to the indications of K. Itakura et al. in Nucleic Acids Research, (1980), 8, 5507.
  • modified nucleic acid groups containing, as base, base X or base Y, which can be used as monomers in the synthesis of the probe precursor according to the invention or which are used in the preparation of dimers or trimers which can be used in this synthesis can be prepared according to known methods for the preparation of monomers usually used in such syntheses, replacing the usual bases for an X or Y group as the case may be.
  • RNA fragments The monomers containing base X or base Y and intended for the preparation of RNA fragments can be prepared in analogy to what is described above.
  • the invention relates to mixed probes as obtained by unblocking the precursors described above, with dismutation, preferably in equal proportions, of the nucleotides containing groups X and / or Y as defined above. 'they contain.
  • the probe according to the invention comprises less than 20 nucleotides per fragment.
  • the invention also includes the probes in which part of the ambiguities have been replaced by one of the nucleotides of the ambiguity, in particular by a nucleotide containing G in the case of a G / A ambiguity and by a nucleotide containing T for a T / C ambiguity. It should be remembered, however, that this choice is likely to introduce "mismatches".
  • the invention also includes the precursors and the mixed probes comprising, in addition to the 2-amino-adenine groups optionally introduced by disproportionation of the group Y, adenine groups modified so that they are capable of giving three. hydrogen bonds with thymine or uracil, in particular 2-amino adenine groups replacing either an adenine group on which there is no ambiguity, or a G / A ambiguity, as explained in more detail in the patent application French filed the same day, by the same applicants, under the title "probes comprising modified adenine groups, their preparation and their uses.
  • the modified adenine groups confer on the hybrid formed by the probe thus obtained better stability due to the increase in its melting point. It is known in fact that when a deoxy-2 'adenosine (A) is replaced in a nucleotide sequence by a 2-amino-deoxy-2' adenosine (A * ), this leads, in the hybrid formed by this sequence, to the transformation of an AT pair having two hydrogen bonds, into an A * T pair having three hydrogen bonds and which has a stability equivalent to that of a CG pair [RC Comtor and PR Schimmel, Biophysical Chemistry, WH Freeman, San Francisco , (1980)].
  • the probe according to the invention when it comprises His modified adenine groups, in particular amino-2 adenine, introduced by dismutaiton Y groups of the precursor and / or replacing at least part of the adenine groups can therefore advantageously ar e used in analysis or extraction techniques, in particular messenger RNA or complementary DNA, calling for evidence involving the formation of immune complexes and possibly involving immunoenzymatic reactions.
  • the probe obtained according to the invention is advantageously marked, with a view to its identification after hybridization, according to a standard technique in the field of biological analyzes.
  • the invention also relates to a probe as defined above, radioactively labeled, in particular by 32 P.
  • the invention relates to the use of the probe defined above in its various aspects in the field of biological analyzes and extractions, in particular of detection and extraction of messenger RNA and complementary DNA, by means of hybridization.
  • amino acid sequence considered is: which corresponds to the mRNA sequence:
  • the organic phase is washed once with 0.2% sodium bicarbonate and then with water until the pH of the aqueous phase is neutral.
  • the organic phase is dried over sodium sulfate, evaporated to dryness and then precipitated with petroleum ether.
  • the product obtained is purified by chromatography on a column of silica eluted with dichloromethane gradually enriched in methanol. 1.54 g of DMT t Tp are obtained (yield 20%).
  • Mass spectrum (spectrum produced with an apparatus marketed under the name VG Micromass 305, in DCI NH 3 at 200 ° C) 303 (72.0) DMT; 245 (1.0) protected phosphate; 178 (2,5) triazole thymine.
  • TLC (dichloromethane / methanol system: 80/20) Rf: 0.575.
  • the product is fluorescent at 310nm.
  • the mixed probe was "mounted” from 19 mg of resin T [resin carrying the nucleotide T and prepared according to the technique of K. Itakura et al. described in Nucleic Acids Research (1980), 8, 5473] with the following trimers:
  • the resin is treated with 500 ⁇ l of a molar mixture of pyridine aldoxime (PAO) in tetramethyl guanidine (TMG) supplemented with 500 ⁇ l of TMG overnight.
  • PEO pyridine aldoxime
  • TMG tetramethyl guanidine
  • HPLC high performance liquid chromatography
  • the precursor of sequence No. 2 is synthesized from 40 mg of resin T, according to the following diagram:
  • the labeled product is purified on a gel (0.4 ⁇ 30 ⁇ 40 cm) containing 19 g of bisacrylamide, 19 g of acrylamide and 10 ml of 1 M tris borate for a total volume of 100 ml.
  • the radioactive band is cut and extracted overnight in 2 ml of water.
  • nucleosides and nucleotides comprising the base Y which can be used in the synthesis of the precursors according to the invention, are new.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP85401666A 1984-08-22 1985-08-21 Sonde mixte et ses applications, son précurseur, leur préparation et dérivés comportant une base modifiée pour cette préparation Withdrawn EP0177382A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8413096A FR2569406A1 (fr) 1984-08-22 1984-08-22 Sonde mixte et ses applications, son precurseur, leur preparation et derives comportant une base modifiee pour cette preparation
FR8413096 1984-08-22

Publications (1)

Publication Number Publication Date
EP0177382A1 true EP0177382A1 (fr) 1986-04-09

Family

ID=9307173

Family Applications (1)

Application Number Title Priority Date Filing Date
EP85401666A Withdrawn EP0177382A1 (fr) 1984-08-22 1985-08-21 Sonde mixte et ses applications, son précurseur, leur préparation et dérivés comportant une base modifiée pour cette préparation

Country Status (7)

Country Link
EP (1) EP0177382A1 (nl)
JP (1) JPS6169789A (nl)
DK (1) DK380185A (nl)
ES (1) ES8701775A1 (nl)
FR (1) FR2569406A1 (nl)
GR (1) GR852028B (nl)
PT (1) PT81010B (nl)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005175A1 (en) * 1991-09-11 1993-03-18 Medical Research Council Improvements in oligonucleotide primers and probes

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2620122B1 (fr) * 1987-09-03 1989-12-29 Pasteur Institut Procede d'obtention par voie chimique d'oligonucleotides marques et applications biologiques

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS TENTH COLLECTIVE INDEX, vol. 86-95, 1977-1981, page 4224CS, Columbus, Ohio, US; "Chemical substances Azanonaborane - Benzene, ethan" *
JOURNAL OF ORGANIC CHEMISTRY, vol. 47, no. 19, 10 septembre 1982, pages 3623-3628, American Chemical Society, US; W.L. SUNG: "Synthesis of 4-(1,2,4-Triazol-l-yl)pyrimidin-2(lH)-one Ribonucleotide and Its Application in Synthesis of Oligoribonucleotides" *
NUCLEIC ACIDS RESEARCH, vol. 9, no. 22, 1981, pages 6139-6151, IRL Press Ltd., Londres, GB; W.L. SUNG: "Synthesis 4-triazolopyrimidinone nucleotide and its application in synthesis of 5-methylcytosinecontaining oligodeoxyribonucleotides" *
TETRAHEDRON LETTERS, vol. 21, no. 23, 1980, pages 2265-2268, Pergamon Press Ltd., Oxford, GB; C.B. REESE et al.: "Reaction between l-arenesulphonyl-3-nitro-1,2,4-triazoles and nucleoside base residues. Elucidation of the nature of side-reactions during oligonucleotide synthesis" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005175A1 (en) * 1991-09-11 1993-03-18 Medical Research Council Improvements in oligonucleotide primers and probes

Also Published As

Publication number Publication date
FR2569406A1 (fr) 1986-02-28
DK380185D0 (da) 1985-08-21
PT81010A (fr) 1985-09-01
PT81010B (fr) 1987-02-06
ES547039A0 (es) 1986-12-16
ES8701775A1 (es) 1986-12-16
JPS6169789A (ja) 1986-04-10
DK380185A (da) 1986-02-23
GR852028B (nl) 1985-12-19

Similar Documents

Publication Publication Date Title
EP0698034B1 (fr) Oligonucleotides phosphorothioates triesters et procede de preparation
EP0191659B1 (fr) Nouveaux nucléosides de dérivés de l' adénosine, leur préparation et leurs applications biologiques
EP0625986B1 (fr) Oligothionucleotides
EP0850949A2 (fr) Nouveaux dérivés utilisables en séquencage d'acides nucléiques
US7321029B2 (en) 2′-arabino-fluorooligonucleotide N3′→P5′ phosphoramidates: their synthesis and use
EP0691978B1 (fr) Copolymere nucleotide(s)/polymere conducteur electronique, son procede de preparation et son utilisation
EP0208599B1 (fr) Nouveaux supports, leur préparation et les intermédiaires obtenus, leur application à la synthèse d'oligonucléotides et les nouveaux nucléosides et oligonucléotides reliés aux supports ainsi obtenus
JPH07502749A (ja) Ｒｎａを調節するための組成物と方法
AU2007315190A1 (en) Click chemistry for the production of reporter molecules
EP0414607B1 (fr) Séquence d'oligonucléotides anti-sens, anti-ARN messager du TNF alpha, procédé de préparation, application à titre de médicaments et compositions pharmaceutiques
EP0176396B1 (fr) Sondes comportant des groupements adénine modifiée, leur préparation et leurs utilisations
EP0607337B1 (fr) Procede de synthese d'acide ribonucleique (arn) utilisant un nouveau reactif de deprotection
EP0177382A1 (fr) Sonde mixte et ses applications, son précurseur, leur préparation et dérivés comportant une base modifiée pour cette préparation
EP0174879B1 (fr) Polynucléotides liés de façon covalente à un support solide, et procédé pour leur obtention
WO1997049833A1 (fr) Procede de formation de complexes d'hybridation dont la stabilite depend peu de la composition en base des deux molecules d'acides nucleiques hybridees
EP0819134B1 (fr) Oligonucleotides a liaison covalente transversale, procede de preparation et synthon utile dans le procede
US5919917A (en) Photocleavable circular oligonucleotides
EP1166119B1 (fr) Methode homogene de detection mettant en jeu un cryptate de terre rare
EP0463043B1 (fr) Procede de stabilisation de l'hybridation de sequences polynucleotiques complementaires
EP0902788B1 (fr) Derives de nucleosides, et leur utilisation pour la synthese d'oligonucleotides
FR2620122A1 (fr) Procede d'obtention par voie chimique d'oligonucleotides marques et applications biologiques
FR2686882A1 (fr) Oligothionucleotides.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE GB IT LI LU NL SE

17P Request for examination filed

Effective date: 19861003

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: ETABLISSEMENT PUBLIC DIT: CENTRE NATIONAL DE LA

Owner name: INSTITUT PASTEUR

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Withdrawal date: 19870616

RIN1 Information on inventor provided before grant (corrected)

Inventor name: IGOLEN, JEAN

Inventor name: DUCHANGE, NATHALIE

Inventor name: ZAKIN, MARIO

Inventor name: LANGLOIS D'ESTAINTOT, BEATRICE

Inventor name: HUYNH-DINH, TAM